Abstract:

An anti-human tenascin monoclonal antibody is described, whose light and
heavy chain variable region sequences are SEQ ID 1 and SEQ ID 2,
respectively, its proteolytic fragments capable of binding to an
antigenic epitope within the region A.sub.(1-4)-D of human tenascin, its
recombinant derivatives, its conjugates and its similar functional
analogues capable of binding to an antigenic epitope within the
A.sub.(1-4)-D region of human tenascin.

Claims:

1. Anti-human tenascin monocloncal antibody, preferably murine, whose
light and heavy chain variable region sequences are SEQ ID 1 and SEQ ID
2, respectively, its proteolytic fragments capable of binding to an
antigenic epitope within the A.sub.(1-4)-D region of human tenascin, its
recombinant derivatives, its conjugates and similar functional analogues
capable of binding to an antigenic epitope within the A.sub.(1-4)-D
region of human tenascin.

2. Fragments of the antibody according to claim 1, optionally containing
additional markers and diagnostic agents.

3. Recombinant derivative of the antibody according to claim 1, in which
the murine constant region is replaced by its human counterpart.

4. Recombinant derivative of the antibody according to claim 1, in which
the murine constant region is replaced by a biologically active
component.

5. Recombinant derivative of the antibody according to claim 1, in which
the murine constant region is replaced by a pharmacologically active
component.

6. Recombinant derivative of the antibody according to claim 1, in which
the murine constant region is replaced by a member of the avidin family.

7. Derivatives of the antibody according to claim 1 conjugated with
biologically active substances.

8. Biotinylated antibody according to claim 1 or biotinylated fragments,
or biotinylated derivatives as defined above.

9. DNA encoding the antibody according to claim 1 or fragments as defined
above.

10. A vector containing the DNA according to claim 9.

11. Host cell containing the vector according to claim 10.

12. Protein coded for by the nucleotide sequences SEQ ID 1 and SEQ ID 2 or
its fragments.

13. DNA encoding the protein or its fragments according to claim 12.

14. Specific CDRs (Complementary Determining Regions) of the antibody
according to claim 1 and proteins containing said CDRs.

15. Hybridoma producing the antibody according to claim 1, deposited at
the Centro di Biotecnologie Avanzate, Largo Rossana Benzi 10,
Genoa--Italy on 12 Nov. 2003 in accordance with the provisions of the
Budapest Treaty, under deposit number PD03003.

16. Process for the preparation of the antibody according to claim 1
comprisinga) immunisation of an animal with the A.sub.(1-4)-D fragment of
human tenascin;b) fusion of somatic spleen cells of said animal with
myeloma cells not producing immunoglobulins;c) selection of the
monoclonal antibody.

17. Use of the antibody or its proteolytic fragments or its recombinant
derivatives or its conjugates or analogues according to claim 1,
optionally biotinylated, or of its fragments, optionally biotinylated, or
of its biotinylated derivatives for the preparation of a pharmaceutical
product useful for the treatment or diagnosis of a disease characterised
by expression of tenascin.

18. Use according to claim 17, in which said disease is a tumour.

19. Use according to claim 18, in which said tumour is selected from the
group consisting of glioma, cancer of the breast, carcinoma of the lung,
fibrosarcoma and squamous-cell carcinoma.

20. Use of the antibody or its proteolytic fragments or its recombinant
derivatives or its conjugates or analogues according to claim 1,
optionally biotinylated, or of its fragments, optionally biotinylated, or
of its biotinylated derivatives for the preparation of a pharmaceutical
product useful for the two-stage perioperative therapy of solid tumours.

21. Pharmaceutical or diagnostic compositions containing an antibody or
its proteolytic fragments or its recombinant derivatives or its
conjugates or analogues according to claim 1, optionally bio-tinylated,
or its fragments, optionally biotinylated, or its biotinylated
derivatives in mixtures with at least one pharmaceutically acceptable
vehicle and/or excipient.

23. Kit for locoregional radioimmunotherapy consisting of 3 vials; vial 1
containing an antibody or its proteolytic fragments or its recombinant
derivatives or its conjugates or analogues according to claim 1,
optionally biotinylated, or its fragments, optionally biotinylated, or
biotinylated derivatives; vial 2 containing avidin; and vial 3 containing
biotin DOTA.

24. Kit according to claim 22, in which, said biotin DOTA in ##STR00003##
vial 5 or vial 3, respectively, is the formula (I) compound in which Q is
a --(CH2)n-- group, where n is a whole number from 4 to 12, in
which case R' is not present, or Q is selected from the group consisting
of --(CH2)a--CH(R')b--(CH2)b--, where a and b
are independently whole numbers from 0 to n, R' is as defined here below,
or Q is cyclohexyl, phenyl, in which case R' is a substituent on the
cyclohexyl or ##STR00004## phenyl ring;R is hydrogen or -.LAMBDA. where
-.LAMBDA. is a formula (II) macrocycle,where the various Y's, which may
be the same or different, are selected from the group consisting of
hydrogen, straight or branched C1-C4 alkyl,
--(CH2)m--COOH, where m is a whole number from 1 to 3, X is
hydrogen, or the group --CH2--U, where U is selected from the group
consisting of methyl, ethyl, and p-aminophenyl, or X is the group
--(CHW)o--Z, where o is a whole number from 1 to 5, W is hydrogen,
methyl or ethyl, Z is a 5- or 6-member heterocyclic group containing one
or more heteroatoms selected from O, N--R1, where R1 is
hydrogen or straight or branched C1-C4 alkyl, and S; or Z is
selected from the group consisting of --NH2,
--NH--C(═NH)--NH2, or --S--R2, where R2 is straight or
branched C1-C4 alkyl;p is the number 2 or 3;R' is selected from
the group consisting of hydrogen, straight or branched C1-C4
alkyl, --(CH2)q-T, in which T is selected from the group
consisting of --S--CH3, --OH, --COOH, and q is the number 1 or 2;R''
has the same meanings as R', upon the following conditions: if R is
-.LAMBDA., R'' is hydrogen; if R is hydrogen, R'' is -.LAMBDA., or R and
R'' are, respectively, --(CH2)r-.LAMBDA.(for R), where r is a
whole number from 4 to 12, and -.LAMBDA.(for R'), Q being a
--(CH2)n-- group, where n is a whole number from 4 to 12.

25. Kit according to claim 22, in which vial 3 contains an avidin dimer in
which two avidin molecules are bound via the --NH2 groups by means
of suberate.

26. Kit according to claim 22, in which said vial 3 contains an avidin
dimer in which two avidin molecules are bound via the --COOH groups by
means of polyethylene glycol with a molecular weight of 3,400.

27. Kit according to claim 22, in which the antibody or its proteolytic
fragments or its recombinant derivatives or its conjugates or analogues,
optionally biotinylated, or its fragments, optionally biotinylated, or
its biotinylated derivatives, are combined with other anti-tenascin
antibodies, preferably targeting the EGF-like region of the protein.

28. Kit according to claim 22, in which the antibody or its proteolytic
fragments, or its recombinant derivatives or its conjugates or analogues,
optionally biotinylated, or its fragments, optionally biotinylated, or
its biotinlyted derivatives, are combined with other tumour-specific
antibodies.

29. Use of the antibody or its proteolytic fragments or its recombinant
derivatives or its conjugates or analogues according to claim 1,
optionally biotinylated, or its fragments, optionally biotinylated, or
its biotinylated derivatives, in the preparation of compositions useful
in the tumour immunolocalisation procedure.

30. Container, preferably in the form of a vial, suitable for injection,
containing an antibody or its proteolytic fragments or its recombinant
derivatives or its conjugates or analogues according to claim 1,
optionally biotinylated, and/or radiolabelled or its fragments according
to claim 2, optionally biotinylated, or its biotinylated derivatives.

31. Tumour imaging method including the administration of an antibody or
its proteolytic fragments or its recombinant derivatives or its
conjugates or analogues according to claim 1, optionally bio-tinylated,
or its fragments, optionally biotinylated, or its biotinylated
derivatives to a person suffering from or suspected of suffering from a
tumour, and the detection of said tumour.

32. Method according to claim 31, in which the antibody or its proteolytic
fragments or recombinant derivatives or conjugates or analogues are
radiolabelled.

33. Combination containing the antibody or its proteolytic fragments or
recombinant derivatives or conjugates or analogues according to claim 1,
or fragments, or derivatives and a second tenascin-specific antibody.

34. Use of the combination according to claim 33 in a sandwich-type
in-vitro ELISA assay, in conditions in which said second antibody binds
to a second antigenic epitope, for the purposes of determining
circulating tenascin levels, particularly levels of the isoforms
containing the A.sub.(1-4)-D region.

Description:

[0001]The present invention relates to anti-human tenascin monoclonal
antibodies, methods and materials for obtaining them, the use of said
antibodies in diagnostic and treatment procedures for tumours expressing
tenascin and materials containing said antibodies suitable for use in the
medical field.

BACKGROUND TO THE INVENTION

[0002]The specificity of tumour therapy often constitutes a limiting
factor in determining the success of the treatment. In fact, the onset of
toxic effects and the reduced tolerability of a number of anticancer
agents limit their use and the quality of life of the patients.

[0003]The reduction of toxicity is directly linked to the selectivity of
the treatment for cancer cells alone. Monoclonal antibodies are the ideal
instrument for specifically localising the tumour and, when combined with
the avidin/biotin amplification system, constitute an extremely potent
method for directing active molecules to the tumour site.

[0004]Tenascin is a molecule of the extracellular matrix, expressed during
embryogenesis and in adult tissues during the processes of scar formation
and tumour development, as well as in newly formed blood vessels.
Tenascin is virtually absent in normal adult tissues, whereas it is
expressed in the stroma of many solid tumours such as gliomas (Burdon, et
al., Cancer. Res., 43: 2796-2805, 1983), carcinomas of the breast
(Chiquet-Ehrismann, et al., 1986), lung (Natali, et al., Intl. J. Cancer,
54: 56-68, 1989), fibrosarcomas and squamous-cell carcinomas (Ramos, D.
M. et al., Intl. J. Cancer, 75: 680-687, 1998). Tenascin is expressed in
glioma, but not in the corresponding normal brain tissue. For an in-depth
discussion of tenascin, the reader is referred to WO 92/04464, Wistar and
related references.

[0006]Other references for this type of cancer treatment are: WO 94/04702,
and U.S. Pat. No. 5,578,287.

[0007]Three-step pre-targeting treatment, also known sotto by the
trade-mark PAGRIT®, is based on the sequential intravenous
administration of a biotinylated anti-tenascin monoclonal antibody,
streptavidin and 90Y-biotin, with administrations of avidin and
biotinylated albumin prior to the streptavidin and 90Y-biotin,
respectively ("chase" step), to reduce the circulating levels of the
antibody and of streptavidin. The selectivity of the 3-step pre-targeting
method is due to the use of the anti-tenascin monoclonal antibody. The
targeting of extracellular matrix molecules presents the advantage,
compared to targeting aimed at cell surface antigens, of not being
affected by modulation of antigen expression by the tumour cell itself.
The doses, administration times and "chases" of the pre-targeting
treatment have been established for the purposes of obtaining optimal
tumour to non-tumour biodistribution ratios.

[0008]The results obtained in 48 patients suffering from glioblastoma
(GBM) or anaplastic astrocytoma (AA), entered into a study by Paganelli
(Paganelli, G., et al., Eur. J. Nucl. Med., 26 (4): 348-357, 1999), have
demonstrated a substantial lack of toxicity, except for a number of cases
of allergic reaction to streptavidin, and preliminary efficacy of the
treatment. In fact, 2 months after the end of the treatment, 25% of the
patients showed a reduction of the tumour mass (Complete Response=6%,
Partial Response=11%, Minor Response=8%), and 52% of patients remained
stable, with an overall response rate of 77%. In a number of these
patients, whose life expectancies were below six months, the response to
treatment persisted for more than one year.

[0009]The role of the biotinylated anti-tenascin antibody is to localise
in the tumour and mediate, via the biotinylated molecule, the
accumulation of avidin and subsequently of 90Y-biotin, directing the
radioisotopes directly into the interior of the tumour. Anti-tenascin
antibodies are already the object of patents and patent applications:
U.S. Pat. No. 5,624,659, Duke University; JP 2219590, Rikagaku; WO
92/04464, Wistar; and WO 03/072608, Sigma-Tau.

[0011]The clone used for the generation of said antibody in the
above-cited studies is called BC-2. The applicant has demonstrated that
the clone BC-2 is not suitable for industrial development, in that it
produces an additional non-functional light chain (probably deriving from
the parent myeloma line) whose level of expression increases during
development of the production process on a large scale, preventing
industrial-scale purification of the antibody.

[0012]There is therefore a perceived need for an anti-tenascin monoclonal
antibody that can be produced on an industrial scale at the purity levels
required for pharmaceutical use.

[0013]The previous patent application WO 03/072608, filed in the name of
Sigma Tau Industrie Farmaceutiche Riunite S.p.A., describes the
generation of the anti-tenascin antibody ST2146, without the additional
non-functional light chain, capable of recognising an antigenic epitope
shared with antibody BC-4, also contaminated by a non-functional light
chain.

[0014]Antibody ST2485, the object of the present invention, recognises an
epitope partly shared with that of BC-2, and therefore located in the
same protein region. This region is greatly expressed in various tumour
tissues. It is therefore important to have a homogeneous monoclonal
antibody which is specific for this region, to be used for cancer
diagnosis or targeting.

[0015]Moreover, the present invention demonstrates that antibody ST2485
presents the advantage of binding to tenascin in addition to antibody
ST2146, thus proving useful in pre-targeting methods involving the
combined use of the two antibodies.

SUMMARY OF THE INVENTION

[0016]An anti-human tenascin monoclonal antibody has now been found which
solves the above-mentioned problems, since it does not possess a
non-functional light chain and presents the advantage of additivity when
used in combination with another anti-tenascin antibody. This antibody is
the object of the present invention, together with the method for
obtaining it and its use in therapy, in particular for the preparation of
a product useful for the treatment of diseases characterised by the
expression of tenascin, such as, for instance, tumours.

[0017]The present invention relates to the antibody and to antibody
fragments, optionally containing additional markers and diagnostic
agents, to processes for obtaining said antibody and antibody fragments,
to pharmaceutical compositions containing said antibody and its fragments
and to diagnostic and therapeutic methods making use of them, as well as
kits useful for implementing said methods.

[0018]The present invention also relates to the DNA encoding said antibody
or fragments thereof, to vectors containing such DNA, to host cells
containing such vectors, to the protein coded for by the nucleotide
sequences SEQ ID NO: 1 and SEQ ID NO: 2, to the DNA encoding the protein
and the fragments, to the specific complementary determining regions
(CDRs) and proteins containing such CDRs. In particular, the object of
the present invention is also the hybridoma that produces said monoclonal
antibody.

[0019]The antibody which is the object of the present invention is
characterised by the sequences of the variable region of the light and
heavy chains SEQ ID NO: 1 and SEQ ID NO: 2, respectively, shown in FIGS.
17 and 18. For the sake of brevity, the antibody which is the object of
the present invention will be identified by the name ST2485. Also
fragments of ST2485 or its chimeric or recombinant derivatives can be
produced and used within the scope of the present invention. Also objects
of the present invention are the proteolytic fragments of said antibody
capable of binding to an antigenic epitope within the A1-4-D region of
human tenascin. In the course of the description of the present invention
what is meant by antibody fragments are those fragments capable of
binding to an antigenic epitope within the A1-4-D region of human
tenascin C.

[0020]According to the present invention, said antibody or proteolytic
fragments are preferably biotinylated.

[0021]Another object of the present invention consists in the hybridoma
cell line, called cST2485, producing the antibody ST2485.

[0022]The hybridoma cell line was deposited at the Centro di Biotecnologie
Avanzate, Largo Rossana Benzi 10, Genoa, Italy, on 12 Nov. 2003 in
accordance with the provisions of the Budapest Treaty, under deposit
number PD03003.

[0023]Also objects of the present invention are the recombinant
derivatives of antibody ST2485, which may optionally be biotinylated. In
particular, the recombinant derivatives preferred are those in which the
murine constant region is replaced by its human counterpart (Ferrer, C.,
et al., J. Biotechnol., 52: 51-60, 1996), or those in which the murine
constant region is replaced by a biologically active component, such as,
for example, a member of the avidin family (Penichet M L., Manuel, L., et
al., J. Immunol., 163: 4421-4426, 1999), a growth factor useful for
stimulating tumour-directed immunological effectors (for example, G-CSF,
GM-CSF), or those in which the murine constant region is replaced by a
pharmacologically active component, such as, for example, a superantigen,
a toxin, a cytokine or any other protein useful for enhancing the
anticancer therapeutic efficacy (Di Massimo, A M, et al., British J.
Cancer, 75 (6): 822-828, 1997; Parente D., et al., Anticancer Research,
17 (644073-4074, 1997). The methods for obtaining such derivatives are
well known to experts in the field.

[0025]In particular, the conjugated derivatives preferred are those in
which the biologically active component is bound to the antibody by means
of conventional systems. Examples of biologically active compounds are
the members of the avidin family, a growth factor useful stimulating
tumour-directed immunological effectors (for example, G-CSF, GM-CSF), or
those in which the murine constant region is replaced by a
pharmacologically active component, such as, for example, a superantigen,
a toxin, a cytokine or any other protein useful for enhancing the
anticancer therapeutic efficacy, anticancer drugs, and radioisotopes.

[0026]A further object of the present invention consists in the use of the
antibody or its derivatives for the preparation of a pharmaceutical
product useful in a method for cancer radioimmunotherapy carried out
preferably according to the method called three-step pre-targeting, also
known by the trade-mark PAGRIT®, including the administration to a
subject suffering from a cancer expressing the tenascin antibody ST2485
or proteolytic fragments thereof, preferably biotinylated.

[0027]Recombinant derivatives of the antibody which is the object of the
present invention and its conjugates can be used to advantage in cancer
therapy. Therefore, the use of the antibody and its fragments or
derivatives in the preparation of a medicine for the treatment of tumours
expressing tenascin constitutes a further object of the present
invention.

[0028]For the purposes of implementing radioimmunotherapy two therapeutic
kits, one systemic and the other locoregional, are also described. These
kits are also known by the trade-mark PAGRIT®. The systemic kit
consists of 5 vials, including vial 1 containing the bio-tinylated
antibody or fragments or derivatives thereof according to the present
invention; vial 2 containing avidin; vial 3 containing streptavidin; vial
4 containing biotinylated human albumin; and vial 5 containing biotin
DOTA (or derivatives of biotin described in WO 02/066075). The
locoregional kits consists of 3 vials, corresponding to vials 1, 2 and 5
of the systemic kit. The vials are prepared so as to be suitable for
injection in the subject to be treated, preferably a human subject. For
the purposes of implementing radioimmunotherapy, a method called IART
(Intraoperative Avidination for Radionuclide Treatment) is described,
based on the intraoperative treatment of patients undergoing surgical
removal of tumour masses with the reagents which are the object of the
present invention, either alone or in combination with other components
of the PAGRIT® kit.

[0029]The specific container, preferably in the form of a vial suitable
for injection containing the antibody or its fragments in biotinylated
form, constitutes a further object of the present invention.

[0030]According to one embodiment of the present invention, in the
therapeutic kits the biotinylated antibody is combined with other
anti-tenascin antibodies, preferably directed at the EGF-like region of
the protein. Alternatively the biotinylated antibody can be coupled to
other antibodies specific to the tumour. A general description of this
type of method is to be found in EP 0 496 074, European Journal of
Nuclear Medicine Vol. 26, No4; April, 1999; 348-357, U.S. Pat. No.
5,968,405.

[0031]Another object of the present invention is the use of the monoclonal
antibody ST2485 for obtaining images for subsequent diagnostic purposes
("imaging") by means of in-vivo immunolocalisation in the tumour.

[0032]Another object of the present invention is the use of the monoclonal
antibody ST2485 in combination with a second anti-tenascin antibody in a
test for the production of a diagnostic kit for determining circulating
levels of tenascin.

[0033]These and other objects of the present invention will be described
in detail here below, also with the aid of examples and figures.

[0035]FIG. 2: SDS-PAGE (a,b) and Western blot (c,d) analysis of antibody
ST2485 in reducing and non-reducing conditions. In reducing conditions, a
double band is observed at the level of the light chain of the antibody
(b,d).

[0036]FIG. 3: SDS-PAGE analysis of antibody ST 2485 subjected to
deglycosylation by digestion with the enzyme PNGaseF
(Peptide-N-Glycosidase from Flavobacterium): disappearance of the high
molecular weight light chain band is observed.

[0044]FIGS. 11 and 11a: biodistribution of ST2485 at various
administration doses compared with BC-2. The accumulation of ST2485 in
the tumour, at all doses administered, is at least twice as much as that
of BC-2.

[0045]FIGS. 12 and 12a: tumour to non-tumour ratios of ST2485 compared
with BC-2. These ratios are higher for antibody ST2485, at all doses
administered.

[0052]The inventors have prepared a new anti-human tenascin antibody
called ST2485, whose light and heavy chain variable region sequences are
SEQ ID NO: 1 and SEQ ID NO: 2, respectively, produced from the hybridoma
cell line cST2485. The antibody is capable of binding to an antigenic
epitope within the A.sub.(1-4)-D region of human tenascin.

[0053]The proteolytic fragments of the antibody according to the present
invention are capable of binding to an antigenic epitope within the
A.sub.(1-4)-D region of human tenascin.

[0054]Recombinant derivatives of the antibody according to the present
invention are obtained according to conventional methods which are well
known to experts in the field. The recombinant derivatives preferred are
those in which the murine constant region is replaced by its human
counterpart, or by a biologically or pharmacologically active component,
or by a member of the avidin family.

[0055]According to the present invention, the conjugated derivatives are
obtained by means of conventional methods well known in this field. The
preferred conjugated derivatives are those in which a biologically active
portion is bound to the antibody. Examples of biologically active
portions are the members of the avidin family, a growth factor useful for
stimulating tumour-directed immunological effectors (such as G-CSF,
GM-CSF), a pharmacologically active portion, such as, for example, a
toxin, superantigen, cytokine or any other protein useful for enhancing
the therapeutic anticancer effect, anticancer drugs, and radioisotopes.

[0056]For further information regarding the preparation of the recombinant
and conjugated derivatives, the reader is referred to WO 03/072608.

[0057]According to the present invention, recombinant or conjugated
derivatives of the monoclonal antibody are also referred to as
"derivatives".

[0058]In one preferred embodiment of the present invention, the antibody,
its fragments and derivatives can be biotinylated.

[0059]The antibody according to the present invention, its fragments and
derivatives can also advantageously contain additional markers and
diagnostic agents.

[0060]The present invention also includes the DNA encoding the monoclonal
antibody or its fragments as defined above. The invention also comprises
a vector containing said DNA, and a host cell containing said vector.

[0061]Vectors and host cells fall within the sphere of the common
knowledge pertaining this field.

[0062]The present invention also comprises the protein coded for by the
nucleotide sequences SEQ ID NO: 1 and SEQ ID NO: 2 or its fragments, and
the DNA encoding it.

[0064]The process for preparing the antibody according to the present
invention consists of the following stages: [0065]a) immunisation of an
animal, preferably a mouse, with the A.sub.(1-4)-D fragment of human
tenascin; [0066]b) fusion of the somatic spleen cells of said animal with
myeloma cells not producing immunoglobulins; [0067]c) selection of the
monoclonal antibody.

[0068]The monoclonal antibody ST2485 is produced from the hybridoma cell
line cST2485, identified above.

[0069]According to the present invention, the antibody or its fragments or
its derivatives, optionally biotinylated, are used for the preparation of
a pharmaceutical product useful for the treatment and diagnosis of
diseases characterised by expression of tenascin. In particular, said
diseases are tumours, and more particularly include glioma, breast
cancer, lung cancer, fibrosarcoma and squamous cell carcinoma.

[0070]In one preferred aspect, the above-mentioned pharmaceutical product
is used in the two-stage perioperative therapy of solid tumours,
described in the International application WO 03/07268. In this type of
therapy, the biotinylated antibody is administered, followed by avidin,
thus constructing the "artificial receptor" for the actual subsequent
anticancer agent. In this case, the anticancer agent will be delivered by
biotin, which will be contained in a chemical compound suitable for
forming a complex with the anticancer agent and hereinafter referred to
as the biotin compound, and will be administered systemically in the
postoperative phase. The biotin, in fact, will localise only where avidin
is present and in this case we can be sure that avidin is present in the
area we are interested in treating, in that it is introduced by the
surgeon a few hours earlier (e.g. 4 to 72 hours) during the operation.
This is an advantage in that it drastically reduces the time elapsing
between removal of the primary tumour and adjuvant therapy.

[0071]As regards the industrial aspects of the present invention, the
antibody described herein will be suitably formulated in pharmaceutical
compositions or therapeutic or diagnostic kits, according to normal
practice in the pharmaceutical field.

[0072]Pharmaceutical compositions and kits are entirely of the type which
is conventional in this field and can be prepared by experts in the field
even only on the basis of common knowledge. Examples of pharmaceutical
compositions are provided in the references cited in the present
invention. The same is true of the kits. Particularly preferred are the
kits for tumour radioimmunotherapy, as described in the above-mentioned
studies by Paganelli et al., and in EP 0 496 074, WO 02/066075, WO
03/072608, and WO 03/075960. A particular use of the radioimmunotherapy
kit can be implemented with the device described in WO 03/069632.

[0073]Pharmaceutical compositions containing the antibody or its
derivatives or fragments, optionally biotinylated in mixtures with at
least one pharmaceutically acceptable excipient or vehicle are included
in the scope of the present invention.

[0075]In another embodiment of the present invention, the kits are for
locoregional radioimmunotherapy and contain 3 vials which are the same as
vials 1, 2 and 5 of the systemic kit.

[0076]In a particularly preferred embodiment, in the vial containing
biotin

##STR00001##

DOTA, said biotin DOTA is the formula (I) compoundin which Q is a
--(CH2)n-- group, where n is a whole number from 4 to 12, in
which case R' is not present, or Q is selected from the group consisting
of --(CH2)a--CH(R')b--(CH2)b--, where a and b
are independently whole numbers from 0 to n, R' is as defined here below,
or Q is cyclohexyl, phenyl, in which case R' is a substituent on the
cyclohexyl or phenyl ring;

##STR00002##

R is hydrogen or -Λ where -Λ is a formula (II)
macrocycle,where the various Y' s, which may be the same or different,
are selected from the group consisting of hydrogen, straight or branched
C1-C4 alkyl, --(CH2)m--COOH, where m is a whole
number from 1 to 3, X is hydrogen, or the group --CH2--U, where U is
selected from the group consisting of methyl, ethyl, and p-aminophenyl,
or X is the group --(CHW)o--Z, where o is a whole number from 1 to
5, W is hydrogen, methyl or ethyl, Z is a 5-, or 6-member heterocyclic
group containing one or more heteroatoms selected from O, N--R1,
where R1 is hydrogen or straight or branched C1-C4 alkyl,
and S; or Z is selected from the group consisting of --NH2,
--NH--C(═NH)--NH2, or --S--R2, where R2 is straight or
branched C1-C4 alkyl;p is the number 2 or 3;R' is selected from
the group consisting of hydrogen, straight or branched C1-C4
alkyl, --(CH2)q-T, in which T is selected from the group
consisting of --S--CHs, --OH, --COOH, and q is the number 1 or 2;R'' has
the same meanings as R', upon the following conditions: if R is
--Λ, R'' is hydrogen; if R is hydrogen, R'' is -Λ, or R and
R'' are, respectively, --(CH2)r-Λ(for R), where r is a
whole number from 4 to 12, and -Λ (for R'), Q being a
--(CH2)n-- group, where n is a whole number from 4 to 12.

[0077]These compounds are described in WO 02/066075.

[0078]In a particularly preferred embodiment, in the vial containing
avidin, the latter is an avidin dimer in which two molecules of avidin
are bound via --NH2 groups by means of suberate, or an avidin dimer
in which two molecules of avidin are bound via --COOH groups by means of
polyethylene glycol with a molecular weight of 3,400, as described in WO
03/075960.

[0079]In the kits according to the invention, the antibody, its
proteolytic fragments, its derivatives, optionally biotinylated, can be
in combination with other anti-tenascin antibodies, preferably targeting
the EGF-like region of the protein, or in combination with other
tumour-specific antibodies.

[0080]All the aspects of this invention, in addition to applying to the
therapy of tumours expressing tenascin, also apply to the diagnosis of
tumours and particularly tumour immunolocalisation procedures, e.g.
imaging. Another object of the present invention is a container,
preferably in the form of a vial suitable for injection, containing an
antibody or its proteolytic fragments or its derivatives, optionally
biotinylated, and/or radiolabelled.

[0081]In another aspect of the invention, the antibody or its fragments,
recombinant derivatives, or analogues are used in combination with a
second tenascin-specific antibody, in a sandwich-type in-vitro ELISA
assay, in conditions in which said second antibody binds to a second
antigenic epitope of tenascin, for the purposes of determining
circulating tenascin levels, and particularly the isoforms containing the
A.sub.(1-4)-D region.

[0084]Production of the ST2485 reference material was achieved by
culturing the cST2485 hybridoma cells in a 2-litre bioreactor; two
successive limiting dilution subclonings of the cST2485 Post Production
Cell Bank (PPCB) led to the selection of the subclone cST2485/A3e/Al2f,
used for the production of the Master Cell Bank (MCB) and the Working
Cell Bank (WCB).

[0085]ST2485 is a mouse immunoglobulin of IgGl/k isotype.

[0086]FIG. 2 shows the SDS-PAGE (a,b) and Western blot (c,d) analysis of
the antibody ST2485 in reducing and non-reducing conditions. In reducing
conditions (b,d), a double band is observed at the level of the light
chain of the antibody; Western blot analysis demonstrates that the two
bands are immunoreactive as light chains of immunoglobulin.
Hydroxyapatite chromatographic analysis of antibody ST2485, shown in FIG.
3, confirms a mild heterogeneity of the antibody through the slightly
asymmetrical peak. In the same analysis, on the other hand, the antibody
BC-2 presents a distinctly heterogeneous profile, in which three peaks
can be distinguished, of which only peak 1 corresponds to the totally
functional homogeneous antibody.

[0087]To define the nature of the heterogeneity of antibody ST 2485, a
sample of the latter was subjected to digestion of the N-glycoside
residues with the enzyme PNGaseF (Peptide-N-glycosidase from
Flavobacterium). The enzyme digestion was done using the Prozyme
deglycosylation kit (cat. No. GE51001), in the conditions indicated by
the manufacturer: approximately 8 μg of ST2485 and 5 mU of enzyme were
reacted overnight at 37° C. in 10 μl of reaction mixture.
Antibody ST2485, digested and undigested, was run on 12% polyacrylamide
gel in reducing conditions. The gel was stained with Coomassie Brilliant
Blue.

[0088]The digestion caused the disappearance of the high molecular weight
light chain band, as demonstrated by the SDS-PAGE analysis in reducing
conditions illustrated in FIG. 4. This finding shows that the two light
chain bands of antibody ST2485 correspond to the glycosylated (high
molecular weight) and non-glycosylated (low molecular weight) variants of
the same. The existence of a potential N-glycosylation site was also
confirmed by the immunoglobulin cDNA sequence data (FIG. 17).

[0090]ST2485 binding to the A.sub.(1-4)-D region of tenascin is specific,
as demonstrated by the Western blot analysis presented in FIG. 5. The
antibody binds to human tenascin and to the A.sub.(1-4)-D fragment, but
not to the EGF-like fragment of the same protein, containing the epitope
recognised by the BC-4 antibody.

[0091]Antibody ST2485 binds human tenascin in a distinct epitope or in one
partly shared with BC-2, as demonstrated by the competitive ELISA assay
between the two antibodies illustrated in FIG. 6. The biotinylated
antibody BC-2, at a concentration established in preliminary experiments,
is dispensed with non-biotinylated BC-2 (curve 1) or ST2485 (curve 2) in
increasing concentrations on plates sensitised with the antigen tenascin
C (a), or Tn A.sub.(1-4)-D fragment (b). The binding of the biotinylated
antibody is measured after addition of HRP-streptavidin and the related
chromogenic substrate TMB. Antibody ST2485 causes 40% inhibition of BC-2
binding to tenascin C or the Tn A.sub.(1-4)-D fragment.

[0092]The immunoreactivity of ST2485 was evaluated in comparison with BC-2
by ELISA assay on tenascin C and on the Tn A.sub.(1-4)-D fragment, as
shown in FIG. 7. The antibodies ST2485 and BC-2 and the normal mouse
immunoglobulins (nMIgG) were dispensed at increasing concentrations on
plates sensitised with tenascin C (a) or with the Tn A.sub.(1-4)-D
fragment (b); the addition of the secondary antimouse antibody labelled
with alkaline phosphatase and the related chromogenic substrate (pNPP)
made it possible to determine the dose-response curve of the antibodies.
The amount of ST2485 necessary to obtain 1.0 OD is approximately 13 times
less than the amount of BC-2 on whole tenascin (a), and approximately 10
times less than the amount of BC-2 on the Tn (A1-4-D) fragment (b).

[0093]The affinity of antibody ST2485 was evaluated both on tenascin C and
on the Tn (A1-4-D) fragment by means of BIAcore analysis. On
tenascin C the KD1 of ST2485 is 9.77×10-10 M
(ka1≈6.02×105;
kd1≈5.88×104), whereas the KD1 of BC-2 is
2.54×10-7 M (ka1=9.85×103;
kd1=2.5×10-3). On the Tn (A1-4-D) fragment the
KD1 of ST2485 is 9.72×10-10 M
(ka1=3.28×105; kd1=3.19×10-4) whereas the
KD1 of BC-2 is 7.39×10-8 M (ka1=2.68×10-4;
kd1=1.93×10-3).

[0094]Maintaining the immunoreactivity after biotinylation constitutes a
fundamental requisite of the antibody used in pre-targeting, and
therefore the behaviour of biotinylated ST2485 (8.3 biotins/molecule) was
evaluated by ELISA and BIAcore assays in comparison with biotinylated
BC-2 (7.6 biotins/molecule). With reference to FIG. 8, the antibodies
ST2485 and BC-2, biotinylated and non-biotinylated, and the normal mouse
immunoglobulins (nMIgG) were dispensed at increasing concentrations on
plates sensitised with tenascin C (a) or with the Tn A.sub.(1-4)-D
fragment (b); the addition of the secondary anti-mouse antibody labelled
with alkaline phosphatase and the related chromogenic substrate (pNPP)
made it possible to determine the dose-response curve of each
biotinylated antibody compared to the non-biotinylated one (residual
immunoreactivity). The residual immunoreactivity of biotinylated ST2485
and BC-2 on tenascin C is equal to 76% and 88%, respectively. On the Tn
A.sub.(1-4)-D fragment, the residual immunoreactivity for ST2485 and BC-2
is 73% and 83%, respectively.

[0095]The BIAcore analysis of the biotinylated antibodies on whole
tenascin shows a KD1 affinity=2.88×10-9 M for
biotinylated ST2485 (ka1=2.8×105;
kd1=8.07×10-4) and a KD1
affinity=3.71×10-7 M for biotinylated BC-2
(ka1=6.0×103; kd1=2.23×10-3).
Biotinylated ST2485 thus maintains good immunoreactivity and affinity
characteristics, conserving an affinity for tenascin approximately 100
times greater than that of biotinylated BC-2.

[0096]Immunohistochemical studies on various human tumours (breast, lung,
stomach, colon) have demonstrated localisation of ST2485 at the level of
the extracellular matrix, similar to that reported for BC-2 (Natali, P G,
et al., Int. J. Cancer, 47 : 811-16, 1991). These studies have also
demonstrated the ability of ST2485 to produce cross-reaction with murine
tenascin, including that expressed in normal mouse tissues, as can be
seen in FIG. 9. Sections of LMM3 tumour (murine mammary carcinoma) and
normal murine intestine fixed in formalin were incubated with 10 μg/ml
of ST2485 or of normal murine IgG1 antibody (control). After incubation
with the biotinylated secondary anti-mouse antibody and the
avidin-biotin-peroxidase complex (Vectastain Elite ABC kit) binding to
tenascin was detected with the addition of the colorimetric substrate DAB
(Vector). The contrast was done with Mayer's haematoxylin. The LMM3 and
murine intestine sections are positive with ST2485 but not with the
control antibody. It was not possible to perform these studies in
comparison with BC-2 since that antibody does not recognise fixed tissue
sections embedded in paraffin. The ability of ST2485 to recognise murine
tenascin, including that expressed by normal tissues, enhances the
significance of the results obtained in the antibody biodistribution
studies in the murine model described here below.

[0097]For the purposes of evaluating the ability of the antibody to
localise in the tumour mass, biodistribution studies of biotinylated
125I-labelled ST2485 and BC-2 were carried out. These studies were
conducted in nude mice which had been implanted with human tumours
expressing tenascin, according to the protocol represented schematically
in FIG. 10.

[0098]The animals were subcutaneously inoculated with 4×106
human colon carcinoma HT-29 cells in 0.1 ml of sterile saline solution.
After 15 days, when the tumour had reached a mass of approximately 200
mg, groups of five animals received intravenous administrations of
125I-BC2, 125I -ST2485, or normal mouse
126I-immunoglobulins (nMIg), all biotinylated (7-9 biotins/mol) and
at five different doses: 0.2-0.5-1-2-5 μg/mouse in 100 μl of
sterile PBS. Five days after administration of the antibodies the animals
were sacrificed and samples of blood, spleen, kidney, liver and tumour
were taken for determining the radioactivity present. Twenty-four hours
prior to the sacrifice the animals received an intravenous administration
of avidin in 100 μl of sterile PBS, at doses 100 times greater than
that of the antibody (chase).

[0099]The results, presented in FIGS. 11 and 11a, show that both BC-2 and
ST2485 localise specifically in the tumour, regardless of the dose (the
amounts of antibody are expressed as percentage of the dose injected per
gram of tissue: % ID/gr). Moreover, ST2485 shows a greater localisation
in the tumour compared with BC-2 at all doses assayed, always maintaining
a higher tumour to non-tumour ratio (FIGS. 12 and 12a). In particular, at
the dose of 1 μg/mouse the accumulation of ST2485 in the tumour is
equal to 16% of the injected dose, whereas the accumulation of BC-2 does
not exceed 5% at any of the doses assayed. At the dose of 1 μg/mouse
the tumour:blood ratio for ST2485 is greater than 20 (optimal dose),
whereas for the antibody BC-2 this ratio never exceeds 6 at any of the
doses assayed. In the other organs, the tumour to non-tumour ratios for
ST2485 at the dose of 1 μg/mouse are 9, 26, and 43 in relation to the
spleen, kidney and liver, respectively. For BC-2, on the other hand,
these ratios are always lower than 7, at all the doses assayed. The
tumour to non-tumour ratios are shown in FIGS. 12 and 12a.

[0100]For the purposes of evaluating the possibility of the combined use
of the anti-tenascin antibodies ST2485 and ST2146 in the PAGRIT® and
TART methods, ST2416 being directed at an epitope localised in the
EGF-like region of the protein (De Santis, R., et al., Br. J. Cancer, 88:
996-1003, 2003, WO 03/072608), in-vitro and in-vivo additivity
experiments were conducted with the two antibodies. FIG. 13, in two ELISA
tests performed, on plates sensitised with tenascin C, shows the absence
of epitope interference between the antibodies ST2485 and ST2146 (13a)
and the additive nature of their binding to the antigen (13b). For the
evaluation of the interference (a) biotinylated antibody ST2485, at
increasing concentrations, was dispensed on microplates sensitised with
tenascin C in the absence (1) and in the presence (2) of ST2146 at
saturating concentration: the superimposed curves 1 and 2 indicate the
absence of epitope interference between the two antibodies, in that the
binding of biotinylated ST2485 to tenascin is not affected by the
presence of ST2146 (and vice versa; data not shown). Curve 3 shows the
signal obtained when seeding biotinylated ST2485 in the presence of
non-biotinylated saturating ST2485 (control).

[0101]FIG. 13b shows the additivity test, in which the biotinylated
antibodies ST2485 and ST2146 were dispensed, in saturating doses,
separately or together, on microplates sensitised with tenascin C: the
mixture of the two antibodies produces a signal equal to the sum of the
signals, thus demonstrating that the binding of the two antibodies is
additive.

[0102]The in-vitro additivity of the two antibodies was also assayed using
BIACore analysis, as shown in FIGS. 14 and 14a. The tenascin was
immobilised on the chip (CM5 sensor chip, Biosense) according to the
method described (De Santis R.; et al.; Br. J. Cancer, 88: 996-1003,
2003). Two consecutive injections of the first antibody were administered
(ST2485 in a, ST2146 in c) at saturating concentrations (10
quadratureM), followed by injection of the second anti-tenascin
antibody (ST2146 in a; ST2485 in c). Sensorgrams a) and c) show that the
two antibodies are capable of binding to the respective epitopes in an
additive manner, as shown by the increase in the resonance signal
produced by each single antibody. Sensorgrams b) and d) represent the
injections of ST2485 and ST2146 respectively, each followed by the
respective isotypical control antibodies, mIgG2b and mIgG1.

[0103]The additivity study of the two antibodies ST2485 and ST2146 was
also conducted in-vivo in the previously described animal model. The
study scheme is illustrated in FIG. 15, whereas the results obtained are
presented in FIG. 16, expressed in ng of antibody per gram of tumour. The
data confirm the additivity of the two antibodies also in the animal
model. The mixture of the two labelled antibodies, in fact, presents an
accumulation in the tumour amounting to 93% of the theoretical value
(mathematical sum of the two individual labelled antibodies). Groups of
control animals were treated with mixtures of radiolabelled antibody and
second "cold" antibody to avoid any interference.

[0106]FIG. 17 shows the SEQ ID NO: 1 sequence of the variable region of
the light chain (VL) of ST2485.

[0107]FIG. 18 shows the SEQ ID NO: 2 sequence of the variable region of
the heavy chain (VH) of ST2485.

[0108]The comparative characterisation of ST2485 compared to BC-2
demonstrates that the antibody ST2485 possesses the following
characteristics: [0109]it recognises an epitope within the
A.sub.(1-4)-D region of human tenascin, close to or partly overlapping
that of BC-2; [0110]it is a homogeneous antibody as regards the
composition of the light and heavy chain, in that the heterogeneity
observed is due to light chain glycosylation variants; [0111]it is more
immunoreactive than BC-2; [0112]it has greater affinity for the antigen
than BC-2; [0113]it is comparable to BC-2 as regards the maintenance of
immunoreactivity after biotinylation; [0114]it is superior to BC-2 as
regards tumour localisation in the animal model; [0115]it binds in an
additive manner with the anti-tenascin monoclonal antibody ST2146 to
tenascin C, both in-vitro and in-vivo in the animal model.